Autoinducers (“AIs”) are extracellular signal compounds used by a variety of bacteria to regulate cellular functions in response to changes in population density. For example, light production by the marine symbiotic bacterium Vibrio fischeri is controlled in a population density-responsive manner by the self-produced, membrane-permeable autoinducer, N-3-oxohexanoyl-L-homoserine lactone (N-3-oxohexanoyl-L-HSL; AI-1). AI-1 accumulates in a population density-dependent manner during bacterial growth. When it reaches a threshold concentration, AI-1, via the autoinducer receptor and transcriptional activator, LuxR, activates transcription of the lux operon, luxICDABEG, which encodes autoinducer synthase (luxI) and luminescence enzymes. (Eberhard et al., Biochemistry 20: 2444-2449 (1981); Engebrecht et al. Cell, 32: 773-781 (1983); Engebrecht et al., Proc. Natl. Acad. Sci. USA 81: 4154-4158 (1984); Hanzelka et al., J. Bacteriol. 177: 815-817 (1995); Shadel et al, J. Bacteriol. 172: 3980-3987 (1990); Slock et al., J. Bacteriol., 172:3974-3979 (1990); Swartzman et al., J. Bacteriol. 172: 6797-6802 (1990)). The autoinduction mechanism in V. fischeri also involves, among other regulatory aspects, an AI-1 mediated luxR negative autoregulation (Dunlap et al., J. Bacteriol. 170: 4040-4046 (1988); Dunlap et al., J. Bacteriol. 171: 3546-3552 (1989); Engebrecht et al., Genet. Eng. 8: 31-44 (1986); Shadel et al., J. Bacteriol. 173: 568-574 (1991); Shadel et al., J. Biol. Chem. 267: 7690-7695 (1992)).
Long thought to be a regulatory mechanism unique to the luminescence system of V. fischeri and certain closely related marine luminous bacteria, autoinduction of gene expression recently has been identified in a wide variety of other bacteria (Fuqua et al., J. Bacteriol. 176:269-275 (1994)). The diversity of species using autoinduction and the chemical and genetic similarities of their autoinduction systems indicate that autoinduction is an evolutionary conserved regulatory mechanism commonly used by bacteria to sense and respond to population density.
All bacteria presently known to utilize AIs associate with higher organisms, i.e., plants and animals, at some point during their lifecycles. For example, Pseudomonas aeruginosa is an opportunistic pathogen in humans with cystic fibrosis. P. aeruginosa regulates various virulence determinants with AI (Davies et al, Science 280: 295 (1998)). Other examples of AI producing bacteria include Erwinia carotovora, Pseudomonas aureofaciens, Yersinia enterocolitica, Vibrio harveyi, and Agrobacterium tumefaciens. E. carotovora infects certain plants and creates enzymes that degrade the plant's cell walls, resulting in what is called “soft rot disease.” E. carotovora produces the autoinducer N-3-oxohexanoyl-L-HSL. Yersinia enterocolitica is a bacterium, which causes gastrointestinal disease in humans and has been reported to produce an autoinducer. P. aureofaciens associates with the roots of plants and produces antibiotics that block fungus growth in the roots. That antibiotic synthesis is under autoinducer control.
In addition to the known naturally occurring autoinducers, recent work has focused on the synthesis and testing of synthetic analogues of certain autoinducers. For example, Bycroft et al., U.S. Pat. No. 5,593,827 have synthesized a series of N-(β-ketocaproyl)-L-homoserine lactone derivatives. The homoserine lactone derivatives are active autoinducers and control gene expression in certain organisms. Additionally, the autoinducer analogue N-(3-oxo-dodecanoyl)-homoserine lactone has been shown to inhibit the activity of P. aeruginosa (Pearson et al., U.S. Pat. No. 5,591,872). Furthermore, autoinducer analogues based on a furanone ring structure have been shown to inhibit homoserine lactone regulated processes in microorganisms (Kjellberg et al., WO 96/29392). Cao and coworkers have synthesized a series of N-acyl homoserine lactones and assessed their binding parameters and structure-function relationship in the V. harveyi lux system. None of these references describes the synthesis of autoinducer analogues that are suitable for attachment to other molecules and surfaces.
Autoinducer molecules are thought to have particular relevance in the progression of cystic fibrosis (CF). CF is the most common inheritable lethal disease among Caucasians. There are approximately 25,000 CF patients in the U.S.A. The frequency of CF in several other countries (e.g., Canada, United Kingdom, Denmark) is high (ranging from 1 in 400 to 1 in 1,600 live births).
Chronic respiratory infections caused by mucoid Pseudornonas aeruginosa are the leading cause of high morbidity and mortality in CF. The initially colonizing P. aeruginosa strains are nonmucoid but in the CF lung they inevitably convert into the mucoid form. The mucoid coating composed of the exopolysaccharide alginate leads to the inability of patients to clear the infection, even under aggressive antibiotic therapies. The emergence of the mucoid form of P. aeruginosa is associated with further disease deterioration and poor prognosis.
The microcolony mode of growth of P. aeruginosa, embedded in exopolysaccharide biofilms in the lungs of CF patients (Lam et al., Infect. Immun. 28: 546 (1980)), among other functions, plays a role in hindering effective opsonization and phagocytosis of P. aeruginosa cells (Pier et al., N. Engl. J. Med. 317: 793-8 (1987); Pier et al., Infect. Immun. 60: 4768-76 (1992)). Although CF patients can produce opsonic antibodies against P. aeruginosa antigens, in most cases phagocytic cells cannot effectively interact with such opsonins (Pressler et al., Clin. Exp. Immunol. 90: 209-14 (1992); Pier et al., Science 249: 537-40 (1990)). Physical hindrance caused by the exopolysaccharide alginate and a functionally important receptor-opsonin mismatch caused by chronic inflammation and proteolysis are contributing factors to the ineffective interactions (Tosi et al., J. Infect. Dis. 162: 156-62 (1990)). Moreover, the biofilm prevents the effective delivery of exogenous antimicrobial agents to the microorganisms of the colony (de Beer et al., Appl. Environ. Microbiol. 60: 4339 (1994)).
Compounds and compositions facilitating the study of autoinduction mechanisms, particularly biofilm formation, and which are effective in disrupting biofilms or retarding their formation would represent a significant advance in the treatment of disease states associated with biofilms, such as CF. Quite surprisingly, the present invention provides such compounds and compositions.